This invention relates generally to the fields of molecular biology and cell biology and, more specifically, to apoptosis and methods for detecting apoptosis in biological samples.
Apoptosis is a process of programmed cell death by which multicellular organisms selectively delete cells. The term necrosis is used to describe the morphological changes undergone by cells that die by processes other than apoptosis. Apoptosis is characterized by a progressive condensation of the chromatin to the inner face of the nuclear membrane, cell shrinkage with consequent loss of membrane contact with neighboring cells, and fragmentation of the cells with formation of membrane-bound acidophilic globules (apoptotic bodies).
The DNA of cells that have undergone apoptosis is cleaved into fragments that are multiples of approximately 180 base pairs. These fragments can be seen after agarose gel electrophoresis as a characteristic xe2x80x9cladderxe2x80x9d develops. This ladder is widely used as a biochemical marker for discriminating apoptotic cell death from necrotic cell death as the DNA of necrotic cells is randomly degraded and does not produce a ladder. The ladder develops as a result of cleavage of nuclear DNA within the linker regions between nucleosomes. Double strand cleavage results from frequent nicks on both DNA strands.
Although the endonuclease responsible for the cleavage of DNA in apoptotic cells has not been definitively identified, candidate nucleases with properties consistent with their involvement in apoptosis have been identified in apoptotic cells.
The endonucleases that have been identified in apoptotic cells are generally similar in their properties to pancreatic DNase I. Specifically, these endonucleases share the following characteristics:
(i) the DNA ends produced by DNase I cleavage (5xe2x80x2-phosphate and 3xe2x80x2-hydroxyl) are the same as those found in apoptotic nuclei;
(ii) DNase I-transfected COS cells show chromatin changes similar to those seen in apoptosis; and
(iii) DNase I cleavage of chromatin produces the same characteristic nucleosomal DNA fragments that can be isolated from apoptotic cells.
Although DNase I has been detected in cells undergoing apoptosis and the tissue distribution of DNase I is consistent with a role in apoptosis, the endonucleases partially purified from apoptotic cells were shown to be distinct from DNase I. There is less evidence for the involvement of DNase II and other endonucleases in apoptosis.
Presently, the terminal deoxynucleotidyl transferase(TdT)-mediated biotinylated dUTP nick end labeling (TUNEL) method is used to detect apoptotic cells in tissue sections. The TUNEL method utilizes TdT to incorporate a biotinylated deoxyuridine label into DNA fragments containing a 3xe2x80x2-hydroxyl group. The label can be detected using a variety of avidin/streptavidin based detection methodologies. Although 3xe2x80x2-hydroxyl groups are present in the double stranded DNA breaks in the apoptotic cells, they are also present in the DNA of cells that have undergone necrotic cell death. This method, therefore, is not suitable for distinguishing between necrotic cell death and apoptotic cell death. In addition, this methodology does not permit the simultaneous detection of 3xe2x80x2-hydroxyl groups and other biologically relevant molecules, such as RNA and proteins.
Presently, there is a need in the art for a methodology to specifically detect apoptotic cells. There is presently no methodology that permits the specific detection of apoptotic cell death without the simultaneous detection of necrotic cells. In the experiments reported here, we determined whether DNA double strand breaks characteristic of those produced by an endonuclease like DNase I can be detected in apoptotic cells in situ.
When DNA is bound to histones or other proteins in chromatin, it is partially protected from the action of endonucleases, which are able to cleave the DNA at approximately 10-bp intervals, the distance of a single helical turn of the DNA. Because of the helical twist of DNA, the two strands are accessible to endonucleases with production of staggered ends as well as some blunt ends. Thus DNase I cleavage of nucleosome-bound DNA gives rise to double strand cuts with 1, 2, or 3 bases of 3xe2x80x2 overhang.
In contrast, DNase II cleavage of DNA in chromatin yields longer 3xe2x80x2-overhangs of an average of 4 bases.
To detect double-stranded DNA ends in apoptotic nuclei in situ, we used several types of double-stranded, labeled DNA fragments which were ligated to DNA ends present in the nuclei of cells in sections of fixed paraffin-embedded tissues. To detect single-base 3xe2x80x2-overhangs, we took advantage of the fact that double-stranded DNA fragments synthesized by Taq DNA polymerase in the polymerase chain reaction have a single 3xe2x80x2 base extension beyond the templated sequence. Although it was originally suggested that Taq polymerase added only deoxyadenosine to the 3xe2x80x2-ends of double-stranded DNA, other work subsequently established that if the last templated 3xe2x80x2-nucleotide synthesized is deoxycytidine, Taq polymerase will add deoxyadenosine or deoxycytidine, leaving no blunt-ended DNA, thus providing a fragment that could potentially ligate to the recessed 5xe2x80x2-base of many of the single-base 3xe2x80x2-overhangs in a random DNA sequence. To prepare a fragment that can be ligated only to blunt ends, PCR was performed using Pfu DNA polymerase, because this polymerase produces blunt-ended products only.
An alternative method of preparing DNA fragments for use as ligation probes is to synthesize oligonucleotides that form ends that can be ligated. Rather than using two complementary strands in the fashion customarily employed for the production of oligonucleotide linkers, a single oligonucleotide may be synthesized such that a region on the 3xe2x80x2-end of the oligonucleotide is complementary to a region on the 5xe2x80x2-end. When the complementary regions of the oligonucleotide anneal, a hairpin structure with a stem and loop spontaneously forms.
The stem structure formed has a double-stranded end that may be ligated to a compatible end. When the regions of complementarity include the 5xe2x80x2-most and 3xe2x80x2-most nucleotides, the stem formed has a blunt end. By adding nucleotides to either the 3xe2x80x2-end or the 5xe2x80x2-end without adding the complementary nucleotide to the other end of the oligonucleotide, an overhang can be created. By including the appropriate number of nucleotides on the desired end, a 3xe2x80x2-overhang or a 5xe2x80x2-overhang of any length can be created. Additionally, any desired sequence can be incorporated into the overhanging portion of the oligonucleotide.
Using the ligation procedure and probes described, we determined that apoptotic nuclei, but not nuclei in necrotic tissue or tissue with other non-apoptotic DNA damage, have DNA ends that can be ligated to labeled DNA fragments with single-base 3xe2x80x2 overhangs. In addition, we found that DNA having ends characteristic of apoptosis could be detected with blunt ended DNA probes as well as with probes having two and three base 3xe2x80x2-overhangs.
In contrast, nuclei with all forms of DNA damage have a high concentration of 3xe2x80x2-hydroxyl DNA ends that are a substrate for terminal deoxynucleotidyl transferase (TdT). As TdT can extend the 3xe2x80x2 base of single-stranded DNA and overhanging, blunt, and recessed 3xe2x80x2 bases of double-stranded DNA, TdT based methodologies are not suitable to distinguish apoptotic cells from necrotic cells. In contrast, ligation based methodologies are suitable to accomplish this very desirable objective.
The present invention provides a methodology for specifically detecting the presence of apoptotic cells in tissue sections. The present invention overcomes the limitations of the prior art by employing a novel ligation methodology to detect DNA fragments that are diagnostic of apoptotic cells.
One aspect of the present invention includes a method for detecting apoptotic cells in tissue sections. This method uses a novel in situ ligation methodology to label double strand DNA breaks with overhanging termini diagnostic of apoptotic cells.
Another aspect of the present invention is to provide a method of detecting and isolating DNA fragments that have defined termini using a solid phase capture assay.
Another aspect of the invention includes a method for detecting the presence of apoptotic cells in a tissue sample using the ligation methodology presented herein in a DNA blot format. The DNA is isolated from the sample and fractionated by size using agarose gel electrophoresis. The DNA is then transferred to a solid support and probed using the ligation methodology. This method is more sensitive than currently available technologies and permits the detection of a small number of apoptotic cells when the apoptotic cells form only a small portion of the tissue sample.
Another aspect of the present invention is to provide a method for detecting the presence of biologically important macromolecules in a cell undergoing apoptosis. These macromolecules include proteins and RNA. This method permits the simultaneous detection of an apoptotic cell and the detection of the presence of specific macromolecules within the apoptotic cell.
Another aspect of the present invention is to provide a methodology for analyzing DNA to determine whether the DNA has been acted upon by a nuclease and, if the DNA has been acted upon by a nuclease, to determine what type of nuclease has acted upon the DNA.
Another aspect of the invention is to provide a method that allows the determination of the nature of DNA damage caused by the activity of nucleases upon the DNA and specific RNA synthesis associated with that damage.